The overall objective of this research program is to identify proteins that regulate the activity or subcellular localization of the alpha subunit (G- alpha) of heterotrimeric G proteins. Regulatory proteins may be proteins whose interaction with G-alpha was not previously appreciated, or they may be novel proteins or protein families. The Regulators of G Protein Signaling (RGS proteins) are a recent example; RGS proteins enhance the intrinsic GTPase activity of many G-alpha subunits, thus tending to decrease the activity of the G proteins. Four proteins that interact with G- alpha-s have been identified in the yeast two-hybrid assay. Two of the four proteins are NEFA and the mu subunit of the clathrin-associated protein AP2. The other two are essentially unknown, one being homologous to a predicted protein of unknown function identified in the C. elegans genome project, and the other described only as an anonymous brain mRNA encoding a 1405 amino acid protein. Subsequent to the identification of NEFA in the two-hybrid assay, we confirmed a physical interaction between NEFA and G-alpha-s. The objective of the present application is to determine the functional significance of this interaction, and to initiate experiments to confirm and characterize the interactions between the other three proteins and G-alpha-s. The first specific aim is driven by the hypothesis that the identification of the proteins NEFA, AP2-mu, C16C10.10, and AB002373 in the two-hybrid assay is due to a physical interaction between each of these proteins and G-alpha-s. 1) The physical interaction between G-alpha-s and proteins identified in the initial yeast two-hybrid screen will be confirmed. The second specific aim is based on the hypothesis that the physical interaction between NEFA and G protein alpha subunits is physiologically relevant. 2) The subcellular and regional distribution of NEFA and its cognate mRNA will be characterized, and the possibility of a functional interaction between NEFA and certain subtypes of G-alpha will be evaluated. The third specific aim is to assess the hypothesis that the existence of a physical interaction between a given target protein and G-alpha-s, as determined in aim l, reflects a functional interaction between the protein and some subtype of G-alpha. 3) Functional consequences of physical interactions, confirmed in aim l, between G-alpha and any of the other three target proteins will be determined.